Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome “fusion-fission” cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P2 to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P 2 from PtdIns(3)P, controls lysosome "fusion-fission" cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4phosphatase that hydrolyzes PtdIns(3,4)P 2 to form PtdIns(3) P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusionfission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3 H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.
Lysosomes are membrane-bound organelles that serve as a cell's main degradative center (1,2). Lysosomes are also key sentinels of cellular nutrient concentration and metabolic activity and have a critical role in initiating diverse signal transduction pathways (3)(4)(5). Lysosomal membranes include diverse phosphoinositides (PtdIns), known to control lysosome function, trafficking, permeability, and general homeostasis through their ability to coordinate recruitment of critical proteins (6). PtdIns exist in seven different forms which are defined by the phosphorylation status of the 3, 4, and/or 5 hydroxyl groups of their inositol head, a process controlled by a number of cellular kinases and phosphatases with PtdIns specificity (7,8).
Of notable importance on lysosomal membranes is phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P 2 ], generated through 5`-phosphorylation of phosphatidylinositol-3monophosphate [PtdIns(3)P] by Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing (PIKfyve) (9,10). Pharmacological or genetic inhibition of PIKfyve and subsequent depletion of PtdIns (3,5)P 2 has been demonstrated to disrupt lysosomal-related processes including autophagic flux, endocytic and phagocytic cargo delivery to lysosomes, substrate export from lysosome, and calcium release (11)(12)(13)(14). Notably, inhibition of PIKfyve also leads to a dramatic enlargement of lysosomes, a phenomenon explained in part by defective membrane recycling from endosomes and/or lysosomes (9,15). PtdIns (3,5)P2 depletion and lysosome enlargement in various cell lines can be effectively induced by the pharmacological PIKfyve inhibitor apilimod (16,17). Specifically, apilimod interacts with the amino acid asparagine (N1939) predicted to be located within the ATP-binding pocket of the catalytic kinase domain of PIKfyve (18). More recently, Choy et al. attributed apilimod-induced lysosome enlargement to the disruption of lysosome fusion-fission cycling and/or disruption of "kiss-and-run", a term which describes a transient membrane fusion event followed by rapid fission event that permits content exchange between lysosomes (11,17,19,20). In their model, PIKfyve inhibition compromises lysosomal fission (the "run" event) and thus promotes lysosome coalescence which results in increased individual lysosome volume and reduced lysosome numbers (17,21).
To better understand a role for INPP4B in lysosome homeostasis, we investigated the consequences of PIKfyve inhibition in Inpp4b +/+ and Inpp4b −/− mouse embryonic fibroblasts (MEFs). Surprisingly, PIKfyve inhibition with apilimod in Inpp4b −/− fibroblasts leads to the formation of very massively enlarged lysosomes, compared to the typically enlarged lysosomes produced in Inpp4b +/+ fibroblasts. Our results suggest that exacerbated lysosomal enlargement was, at least in part, a result of aberrantly elevated levels of lysosomal PtdIns(3)P generated by increased activity of the Class III PtdIns 3-kinase VPS34 in Inpp4b −/− cells. Together, these point to a novel role for Inpp4b in suppressing VPS34 activity and the existence of coordinated functions for Inpp4b, PIKfyve, and VPS34 in maintaining normal lysosomal homeostasis, dynamics, and function.
To confirm that the aberrant vacuolation response upon apilimod treatment was indeed a result of Inpp4b deficiency, we transduced Inpp4b −/− MEF with constructs that express Inpp4b-EGFP, catalytically dead Inpp4b C845A -EGFP, and control EGFP and treated with apilimod or vehicle and measured vacuole size and number (Fig. S1, G-I). Inpp4b-EGFP expression in Inpp4b −/− MEF generated significantly reduced numbers and average size of enlarged vacuoles upon apilimod treatment compared to nontransduced and EGFP negative controls (Fig. S1, G-I). Notably, transduction of Inpp4b C845A -EGFP in Inpp4b −/− MEF was unable to rescue the number and size of enlarged vacuoles upon apilimod treatment (Fig. S1, G-I). Exacerbated lysosomal enlargement observed with INPP4B deficiency was also generalizable to human cells, as determined in U2OS cells upon INPP4B knockdown with RNAi (Fig. S2, A-E). We also evaluated the effect of Inpp4b suppression on late endosomes. Inpp4b −/− MEF displayed reduced CD63 + late endosome puncta number (Fig. S3). Notably, apilimod treatment did not alter CD63 + late endosome levels, indicating that late endosomes do not reflect the changes observed in lysosomal levels. This suggests that CD63 + late endosomes are not affected by apilimod, and that PIKfyve inhibition may selectively disrupt lysosomal homeostasis and dynamics (Fig. S3). Overall, apilimod treatment in Inpp4b −/− MEF demonstrate an exacerbated lysosomal vacuolation phenotype compared to Inpp4b +/+ MEF that can be rescued by WT Inpp4b, but not a catalytically dead Inpp4b. These data suggest an important role for Inpp4b phosphatase function in maintaining lysosomal dynamics and homeostasis.

Inpp4b deficiency exacerbates apilimod-mediated inhibition of endocytic trafficking to lysosomes
We attempted to measure lysosome dynamics using Lucifer yellow (LY), a membrane impermeable fluorescent dye that is internalized by endocytosis and accumulates within lysosomes. LY fluorescence provides an effective tool to specifically delineate lysosomes for accurate measurement of lysosome number and volume (30,31). MEF were treated with vehicle or apilimod prior to LY pulses of 1, 2, and 4 h. Flow cytometry was used to measure LY uptake which demonstrated that apilimod elevated total intracellular LY-fluorescence similarly in both Inpp4b +/+ and Inpp4b −/− MEF for up to 4 h (Fig. S4). To monitor specific LY trafficking to lysosomes, we labeled lysosomes with ectopic expression of LAMP1-mCherry and measured colocalization with LY (Fig. S5A). Apilimod treatment demonstrated a markedly reduced accumulation of LY to lysosomes in Inpp4b +/+ MEF.
Comparatively, Inpp4b −/− MEF has significantly less LY accumulation at lysosomes compared to Inpp4b +/+ MEF for up to 2 h, indicating a slower colocalization of LY with lysosomes with Inpp4b deficiency (Fig. S5, A and B). We performed the same assay but replaced LY with DQ-BSA, bovine serum albumin labeled with a self-quenched BODIPY TR-X dye that upon lysosomal delivery is cleaved by lysosomal hydrolases, resulting in bright green fluorescent signal (32,33). We observed that DQ-BSA activation was delayed but not inhibited in apilimod-treated Inpp4b −/− MEF (Fig. S5C), suggesting that degradation of lysosomal cargo was not compromised. These findings indicate that PIKfyve inhibition reduces the ability of endocytosed cargo to reach terminal lysosomes, with no major effects on lysosomal proteolysis. Interestingly, like the vacuolation phenotype, apilimod-mediated accumulation was exacerbated in Inpp4b −/− MEF, further indicating a role for Inpp4b in lysosomal trafficking (11,34).

Inpp4b deficiency exacerbates apilimod-mediated lysosome fusion-fission dynamics
Given that apilimod blocks lysosomal accumulation of endocytic cargo such as LY, we adjusted our treatment conditions so that MEF were first pulsed with LY followed by acute treatment with apilimod ( Fig. 2A). In these experiments, quantitation of LY-labeled lysosomes revealed that individual basal lysosome size was similar in Inpp4b +/+ or Inpp4b −/− MEF. Apilimod induced significant lysosomal enlargement compared to vehicle, and lysosome enlargement in Inpp4b −/− MEF was significantly larger than Inpp4b +/+ MEF (Fig. 2, A and B). Inpp4b −/− MEF have fewer total lysosomes than Inpp4b +/+ MEF; and apilimod treatment reduced lysosome numbers in both Inpp4b +/+ or Inpp4b −/− MEF (Fig. 2, A and C). Notably, total lysosomal content as measured by total cellular LY accumulation was significantly lower in Inpp4b −/− MEF and remained unchanged upon apilimod treatment (Fig. 2, A and D). These data conform to a model whereby apilimod treatment promotes lysosomal coalescence, which is manifested as increased individual lysosome volume and reduced total lysosome numbers (16,17,35). Notably, Inpp4b deficiency exacerbates apilimod-induced lysosomal enlargement, thus providing further evidence of a role for Inpp4b in lysosomal homeostasis and dynamics which is revealed upon apilimod treatment. Inhibition of lysosomal fission has been proposed as an explanation for apilimod-induced lysosome enlargement (16,17,35). To investigate the specific consequence of Inpp4b deficiency on "kiss-and-run" and/or "fusion-and-fission" cycles, live-imaging of LY-labeled lysosomes was recorded. Inpp4b +/+ and Inpp4b −/− MEF treated with either vehicle or apilimod were monitored for up to 15 min and lysosomal fission events were recorded. Despite presenting similar fission rates in vehicle-treated Inpp4b +/+ and Inpp4b −/− MEF, apilimod significantly reduced lysosome fission rates in Inpp4b +/+ MEF, and remarkably fission was nearly abrogated in Inpp4b −/ − MEF (Fig. 3, A and B and Movies S1-S4). This suggests that the enhanced lysosomal coalescence observed in Inpp4b −/− MEF upon apilimod treatment is due to an exacerbated inhibition of lysosomal fission and Inpp4b deficiency alters lysosome dynamics such that they are sensitized to the lysosomal fission-inhibiting effects of apilimod. To quantify the differential effects of apilimod on lysosomal proteolytic function, we used the membrane permeable cathepsin B substrate-Magic Red which upon hydrolysis forms membrane impermeable fluorescent cresyl-violet and accumulates within functional lysosomes (37). The results of this assay paralleled the transcript and protein expression levels of cathepsin B, where apilimod treatment had no effect in Inpp4b +/+ MEF, and in Inpp4b −/− MEF, despite having a reduced steady state cathepsin B activity, apilimod treatment   led to a more than 3-fold induction of cathepsin B activity (Fig. 4, F and G). Although activation of nuclear translocation of TFEB by apilimod was no different in Inpp4b +/+ and Inpp4b −/− MEF, lysosomal gene expression, protein expression, and proteolytic function of lysosomes are differentially impacted by apilimod treatment in Inpp4b +/+ and Inpp4b −/− MEF.

Inhibition of autophagy by apilimod is potentiated in Inpp4bdeficient cells
PIKfyve inhibition has been demonstrated to inhibit autophagy in various physiological and pathological settings upon extended PIKfyve inhibition, but not upon acute exposure (12,16,38). We evaluated how apilimod treatment affected autophagy in the context of Inpp4b deficiency by first measuring levels of MAP1LC3A/B (LC3), a membrane protein specifically expressed on autophagosomes by IF with anti-LC3 antibodies (39). Total cellular LC3 staining was similar in vehicle-treated MEF. Apilimod treatment induced LC3 levels to significantly higher levels in Inpp4b −/− MEF than in Inpp4b +/+ MEF (Fig. 5,  A and B). Notably, the corresponding elevation in autophagosome levels induced by apilimod in Inpp4b −/− MEF was fully rescued by ectopic INPP4B expression, indicating that LC3 induction levels are restricted by Inpp4b (Fig. S6, A-C). We also assessed extent of autolysosome formation by quantitating colocalization LAMP1-mCherry fluorescence and LC-3 by IF. Relative to vehicle-treated MEF, colocalization of LAMP1 (lysosomes) and LC3 (autophagosomes) was elevated upon apilimod treatment, and Inpp4b −/− MEF had significantly higher levels of colocalization than Inpp4b +/+ MEF (Fig. 5, A and C). Autophagy was also assessed by measuring LC-3 status by immunoblotting (40). During autophagy, cytosolic LC3-I (16 kDa) is conjugated with phosphatidylethanolamine on the phagophore membrane to form LC3-II (14 kDa). LC3-II is absent in vehicle-treated Inpp4b +/+ and Inpp4b −/− MEF  Autophagy was further assessed by estimating autophagic flux using a reporter system which expresses a fusion of LC3 protein with the acid-insensitive mCherry protein and the acid-sensitive GFP protein (41,42). This reporter permits the estimation of autophagic flux-autophagosome to autolysosome transition-by measuring the ratio of yellow (mCherry + GFP + ) or red (mCherry + GFP -) puncta, respectively (12,43,44). In vehicle-treated Inpp4b +/+ and Inpp4b −/− MEF, we observed predominantly red fluorescence indicating functional transition through autophagy (Fig. 5, F and G). By contrast, apilimod treatment resulted in predominantly yellow puncta in 150 Inpp4b +/+ MEF and an even higher ratio of yellow to red puncta in Inpp4b −/− MEF. (Fig. 5, F and G). Notably, the accumulation of yellow fluorescence (mCherry + GFP + ) in apilimod-treated Inpp4b −/− MEF appear to be on the membranes of enlarged vacuoles. These data demonstrate that Inpp4b deficiency alone does not alter autophagic flux, however, apilimod treatment reveals that Inpp4b regulates autophagic flux in some conditions, including PIKfyve inhibition.

Apilimod-mediated lysosome enlargement is driven by VPS34mediated PtdIns(3)P production
We sought to gain a further understanding of the mechanisms leading to elevated cellular PtdIns(3)P and its role in the exacerbated lysosomal enlargement upon apilimod treatment in Inpp4b −/− MEF. Given its major role in cellular PtdIns(3)P generation, we explored VPS34 functions (45). No changes in VPS34 protein levels were observed by immunoblot in Inpp4b deficiency nor in vehicle-or apilimod-treated conditions (Fig. 7A). Surprisingly, measurement of the lipid kinase activity of VPS34 demonstrated a significant elevation only in apilimod-treated Inpp4b −/− MEF (Fig. 7B), thereby explaining elevated PtdIns(3)P levels.
To elucidate a role for PtdIns(3)P in the formation of massively enlarged vacuoles in apilimod-treated Inpp4b −/− MEF, we reasoned that depletion of cellular PtdIns(3)P could attenuate this phenotype. Thus, we treated MEF with both apilimod and the specific VPS34 inhibitor, VPS34-IN1 (25,46). PtdIns(3)P IF performed on VPS34-IN1-treated MEF demonstrated that VPS34 inhibition can effectively diminish total cellular and lysosomal localized PtdIns(3)P levels (Fig. 7, C-E). Notably, VPS34 inhibition in apilimod-treated Inpp4b −/ − MEF rescued the exacerbated lysosome enlargement but only to levels of enlargement normally observed in Inpp4b +/+ MEF;   EDITORS' PICK: PIKfyve inhibition in Inpp4b-deficient MEF VPS34-IN1 had little effect on the enlarged lysosome size and number in apilimod-treated Inpp4b +/+ MEF, indicating that induction of PtdIns(3)P levels may not be necessary or sufficient for normal apilimod responses (Fig. 7, F-H). In sum, VPS34-IN1 revealed that aberrant hyperactivation of VPS34 in Inpp4b −/− MEF treated with apilimod leads to exacerbated lysosomal enlargement. These findings suggest the existence of a role for Inpp4b in the regulation of VPS34 activation, and thereby PtdIns(3)P levels, in cells undergoing lysosomal stress induced by PIKfyve inhibition.

Discussion
Lysosomes, the primary catabolic organelles in the cell, play pivotal roles in many cellular processes including cell differentiation, plasma membrane repair, programmed cell death, nutrient sensing, and stress responses (47). Thus, exquisite control of lysosome homeostasis, including the dynamic mechanisms that control total lysosomal content, number, and size are critical to maintaining normal lysosomal and cellular functions. Emerging data indicates key roles for PtdIns in maintaining lysosomal homeostasis. In this study, we explored the dynamics of PIKfyve inhibition and resultant PtdIns(3,5)P 2 depletion, which leads to disruption of lysosomal "fusionfission" cycles by compromising "fission", thereby generating enlarged coalescent lysosomes (11,16,17). PIKfyve inhibition also disrupts endocytic cargo delivery, autophagosome formation, and autophagic flux, possibly a result of lysosome membrane perturbations where lysosomes fail to resolve from other lysosomes, late endosomes, and autophagosomes (11,12). PIKfyve has been shown to functionally coordinate with the Class III PI3-Kinase VPS34 through its catalytic product PtdIns(3)P, which serves as a membrane localization target for PIKfyve, as well as a substrate precursor for PtdIns(3,5)P 2 synthesis (45). Together, the functions of PIKfyve and VPS34 recruit and regulate components of endosomal and lysosomal recycling (48). This PIKfyve-VPS34 crosstalk suggests the existence of other PIKfyve cross talks with other PtdInsmodifying enzymes.
Emerging studies present a role for the lipid-phosphatase INPP4B in the regulation of lysosome-associated functions. Our group has demonstrated that INPP4B expression in acute myeloid leukemia regulates lysosomal biogenesis and functions which are crucial for leukemia stem cell maintenance, differentiation, and chemoresistance (29). Another study demonstrated that INPP4B depletion in triple negative breast cancer resulted in delayed EGFR trafficking from early endosomes to late endosomes/lysosomes (49). A role for INPP4B in lysosomal functions is further supported by the observation that elevated expression of INPP4B in PIK3CA-mutant ER + breast cancer cells induce formation of late endosomes and lysosomes, increase cargo trafficking toward lysosomes, and promote endosomal sequestration and lysosomal degradation of key signaling proteins (28). Together, these data support a role for INPP4B in the enhancement of function and cellular content of lysosomes. Indeed, this study further supports this notion as Inpp4b-deficient MEF consistently demonstrated reduced total lysosomal content and lysosome numbers as measured by LAMP1 IF, lysotracker labeling, and lysosomal accumulation of LY (Figs. 1, 2 and S1). Furthermore, lysosomal transcript and protein expression levels were reduced in Inpp4b −/− MEF, together suggesting a role for Inpp4b in promoting and/or maintaining lysosomal content by controlling biogenesis through transcriptional mechanisms (Fig. 4). Since INPP4B and PIKfyve both regulate lysosome function, we sought to shed light on putative interactions between these two enzymes.
Apilimod-mediated PIKfyve inhibition in MEF leads to formation of numerous enlarged translucent cytoplasmic vacuoles readily seen by light microscopy. In previous studies, 20 nM apilimod for 1 h in RAW macrophages led to rapid lysosome enlargement to a volume of 10 μm 3 . HeLa or RPE cells required 100 to 200 nM apilimod to generate lysosome of 3 to 4 um 3 (16,17). By comparison, apilimod treatment of Inpp4b +/+ MEF at 10 nM or 500 nM for 1 h dilated lysosome volume to 3 um 3 or 4.5 um 3 , respectively. The key findings made in this study stem from the observations that apilimod treatment of Inpp4b −/− MEF generate significantly larger cytoplasmic vacuoles than apilimod-treated Inpp4b +/+ . This observation was the first indication of a putative crosstalk between INPP4B and PIKfyve. To gain further insight into the exacerbated vacuolation phenotype in Inpp4b-deficient MEF, we first confirmed that enlarged vacuoles were of lysosomal origin as proposed by Choy et al. (17). Loading of MEF with LY prior to apilimod stimulation permitted effective lysosomal accumulation, which then enabled the clear visualization and quantitation of lysosomal features after apilimod treatment. LY labeling confirmed that lysosome size and number were affected to a greater degree in Inpp4b −/− MEF by apilimod; and that apilimod inhibits lysosomal accumulation of cargo, a phenotype which was also exacerbated in Inpp4b −/− MEF. Lysosome dynamics in Inpp4b −/− MEF were exquisitely sensitized to apilimod, as demonstrated by the severely compromised lysosomal fission, relative to the moderately compromised fission rates observed in Inpp4b +/+ MEF.
In our attempts to further understand the underlying biology responsible for the exaggerated response to apilimod observed in Inpp4b −/− MEF, we observed that the expression of representative lysosomal gene transcripts were either unchanged or decreased in Inpp4b +/+ MEF treated with apilimod. Inpp4b −/− MEF on the other hand demonstrated a significant 2-3-fold increase in the expression of all lysosomal transcripts tested. Elevated lysosomal biogenesis observed in Inpp4b −/− MEF was not explained by the nuclear localization of Tfeb; however, altered Tfeb transactivation activity by phosphorylation and acetylation by the action of factors including mTORC1, PKC, PKD, GSK3β, and phosphatases such as the Ca2+-dependent calcineurin cannot be formally excluded with our data (50)(51)(52). Analysis of lysosomal protein levels showed that lysosomal membrane-embedded proteins including Lamp1 and V-ATPase (V1H) subunit were significantly elevated in Inpp4b +/+ and Inpp4b −/− MEF upon apilimod treatment. On the other hand, cathepsin-B protein levels and activity are elevated only in apilimod-treated Inpp4b −/− MEF, suggesting that the stability of some lysosomal proteins may also be differentially regulated in apilimod-treated Inpp4b +/+ and Inpp4b −/− MEF. Disruption of autophagic flux, which blocks turnover of lysosomal proteins (12,38) may explain elevation of some lysosomal proteins (LAMP1 and V-ATPase). Also, it is currently unclear if elevated cathepsin-B protein and activity result due to disrupted feedback regulation from apilimod effects. In sum, the specific consequences of Inpp4b deficiency combined with PIKfyve inhibition on gene expression require further study.
Our data also show that apilimod can elevate autophagosome levels and promote colocalization with lysosomes to form autolysosomes, however, autolysosome maturation appears to be inhibited as indicated by reduced autophagic flux. Interestingly, enlarged lysosomes in apilimod-treated cells are proteolytically competent as measured by Magic Red and DQ-BSA suggesting that altered lysosomal membrane composition may confer resistance to membrane degradation or other membrane-associated processes required for autolysosome maturation. Our observations are consistent with previous studies regarding the consequences of apilimod (12,(53)(54)(55), with the exception that Inpp4b −/− MEF display exacerbated versions of this phenotype.
Finally, we measured intracellular PtdIns levels in apilimodtreated MEF. PtdIns(3)P levels were observed to be unchanged in vehicle-treated MEF, which suggests that other major mechanisms govern PtdIns(3)P levels, and Inpp4b may only be a minor contributor. As previously reported, apilimod leads to moderately elevated levels of PtdIns(3)P, which we also observed in Inpp4b +/+ MEF (56). Surprisingly, apilimod treatment in Inpp4b −/− MEF led to a dramatic increase in both total cellular and lysosomal levels of PtdIns(3)P. This observation was deemed paradoxical given that Inpp4b deficiency should generate less PtdIns(3)P and suggests that Inpp4b may regulate PtdIns(3)P levels through an indirect mechanism. To explain elevated PtdIns(3)P, our tests revealed that VPS34 activity, but not protein levels, was elevated. Furthermore, VPS34-IN1, a specific inhibitor of VPS34, depleted PtdIns(3)P and reversed the exacerbated lysosomal enlargement observed with Inpp4b deficiency. These data point to a direct role for VPS34 in apilimod-mediated PtdIns(3)P induction (Fig. 8). Furthermore, our results highlight a novel regulatory signaling axis linking PIKfyve, VPS34, and Inpp4b functions.
In conclusion, our study demonstrates that Inpp4b deficiency sensitizes cells and lysosomes to the plethora of effects conferred by apilimod-mediated PIKfyve inhibition, the most obvious of which is significantly exacerbated lysosomal enlargement compared to WT cells, most likely due to blocked fission and thus, enhanced lysosome coalescence. Similar exacerbated consequences are observed for various other phenotypes associated with PIKfyve inhibition including disrupted autophagy and reduced cargo delivery to lysosomes (11)(12)(13)(14). Finally, we have uncovered a novel role for Inpp4b in regulating the activation of VPS34 activity and induction of PtdIns(3)P levels, which was only revealed upon lysosomal stress activated by PIKfyve inhibition. This function is mediated through a novel PIKfyve-VPS34-Inpp4b regulatory signaling axis.

Retroviral transduction
3.0 × 10 6 HEK 293T cells were grown in a 10 cm dish for 24 followed by calcium phosphate transfection. Briefly, 10 μg of retroviral plasmid pWZL hygro SV40 T-Large was mixed with 5 μg of pCL-Eco retroviral packaging vector and 2M CaCl 2 to a final volume of 300 μl in sterile water. The transfection mix was supplemented with equal volume of 2× Hepes-buffered saline (140 mM NaCl, 1.5 mM Na 2 HPO 4 ) followed by addition to the cells. Following 24 h post transfection, the media was changed and supplemented with complete DMEM growth media. Media was collected 48 h and 72 h post transfection. Virus-enriched media was filtered through a 0.45-micron filter, supplemented with 8 μg/ml protamine sulfate, and added to MEF cells grown in 10 cm dishes. Infections were repeated every 8 h and MEF cells were selected for 4 days with 75 μg/ml hygromycin B.

Lysosome labeling
MEF cell lysosomes were labeled with 1 mg/ml LY (Thermo Fisher Scientific) for 2 h in complete growth media at 37 C and 5% CO 2 , followed by washing in PBS and supplementation of complete media for 1 h. LTR (Thermo Fisher) was also used to label MEF cell lysosomes by incubation at 1 μM for 30 min in complete growth media. Magic Red (Abcam) was used to assess lysosomal cathepsin B activity in MEF cells by incubation for 1 h according to manufacturer instructions.

Live-and fixed-cell microscopy
Manual assessment of vacuole and lysosome size, number, LAMP1 positive vesicles, TFEB-GFP localization, Magic Red, or LTR-stained lysosomes were performed using EVOS-FL fluorescent inverted microscope controlled by EVOS XL core imaging system at 20× 0.4 NA. (Thermo Fisher) objective. Spinning disc confocal microscopy was used to perform live imaging through Olympus IX81 inverted microscope connected to Hamamatsu C9100-13 EMCCD camera with 60× 1.35 NA. objective and controlled by Volocity 6.3.0 (Perki-nElmer). Time lapse live imaging performed with an environmental chamber set to 37 C and 5% CO 2 in DMEM complete media. Fixed cells were observed through ZEISS AxioImager M2 Epifluorescence microscope connected to AxioCam MRm CCD camera and controlled by AxioVision Software Version 4.8 at 20× 0.8 NA. or 40× 1.4 NA. objective (Carl Zeiss).

Image analysis
To identify vacuoles as LAMP-1+ and of lysosomal origin, first, we set a minimum size threshold exclusion parameter to identify vacuoles as being greater than 1.5 μm in diameter on light microscopy using ImageJ. LAMP-1-mCherry fluorescence was then overlaid on these enlarged vacuoles and percentage LAMP-1-mCherry positivity was calculated. To measure the percentage of cells with nuclear TFEB, cells were scored to have nuclear TFEB if the nucleus had greater intensity than cytosol using ImageJ. To measure the percentage of lysosomes filled with LY per cell, images imported into ImageJ and number of LAMP1-positive vesicles were manually scored for presence of LY signal within the lysosome lumen.
To quantify LAMP1 or LC3 immunostaining through ImageJ, intensity thresholding was applied to identify fluorescent structures and the mean intensity was obtained for each cell. To quantify LC3 intensity over LAMP1-positive structures, ImageJ was used to threshold for LAMP1-mCherry signal and generating a mask, which was applied to the green (LC3 immunostain) channel to measure LC3 intensity on LAMP1-mCherry positive regions. For MEF cells transiently expressing mCherry-eGFP-LC3B, similar approach was used to determine LC3 green puncta intensity over LC3 red puncta structures where relative intensity ratio greater than 1 indicate formation of autolysosomes due to reduced autophagic flux. Similar image analysis technique was applied for MEF cells transiently expressing LAMP1-mCherry to evaluate PtdIns(3,4)P 2 or PtdIns(3)P levels overlayed on LAMP1-mCherry positive regions within a cell. To quantify CD63 puncta number per cell, intensity thresholding was similarly applied to identify fluorescent puncta structures followed by total CD63 puncta number per cell.
To analyze lysosome-to-cytosol intensity ratio of INPP4B-mCherry, images were imported into ImageJ, and lines were assigned to nucleus-excluded areas of a cell measuring 40-pixel in length and 5-pixel in width. Intensity plot profiles were acquired and exported into excel spreadsheet. Intensity arranged according to values and ratio obtained for highest 10 pixels over lowest 10 pixels (F H /F L fluorescent ratio), where ratio values of approximately 1 represent cytosolic distribution.
To measure lysosome volume and number per cell, particle detection and volumetric tools from Volocity 6.3.0 were used. Briefly, Z-stack images imported into Volocity and punctate lysosome structures identified by applying a 2× cytosol intensity threshold to exclude cytosol and background. Further criteria to include particles greater than 0.3 μm 3 removed noise-derived particles. Each cell was isolated by drawing region interest for individual cell analysis. Quantification of lysosome splitting frequency was performed through Imaris (BitPlane) using 'ImarisTrackLineage' module, where lysosome splitting was defined as frequency of events where two particles were produced from a single particle.

VPS34 immunoprecipitation and VPS34 kinase assay
Following vehicle or apilimod treatment of Inpp4b +/+ and Inpp4b −/− MEF cells, VPS34 immunoprecipitation was performed using the protocol from Cell Signaling (#4263 from Cell Signaling). Five hundred microgram of total protein lysate was incubated with VPS34 antibody #4263 (1:50) overnight at 4 C. Precleared protein A magnetic beads (Cell Signaling) was incubated with immunocomplex for 20 min at room temperature on a shaking rotator. The magnetic beads were washed five times with TBST buffer using DynaMag-2 Spin Magnet (Thermo Fisher). Kinase assay performed on immunoprecipitated VPS34 using Class III PI3K ELISA Kit (K-3000, Echelon) through the protocol recommended for beads conjugated to enzyme. Kinase assay and PI3P production was performed at 30 C for 1 h at 1100 r.p.m. Solution-containing PI3P was isolated from the magnetic beads using DynaMag-2 Spin Magnet and used for colorimetric detection of PI3P on ELISA plates.

Generation of NeuroMab Inpp4b antibody
Hybridoma cells that secrete NeuroMab clone N171/17anti-INPP4b antibody were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillinstreptomycin (Gibco). Cells were passaged every 2 days up until 7 days before collection of antibody containing media by centrifugation at 200g. The antibody-containing media was directly applied to nitrocellulose membrane for Inpp4b detection.

Flow cytometry
MEF cells were incubated with 10 μg/ml DQ-BSA (Invitrogen) or 1 mg/ml LY (Thermo Fisher) for 1 h to 6 h at 37 C, or LTR 1 μM for 30 min, or Magic red for 1 h. Alternatively, LAMP1-mCherry signal of U2OS cells are recorded through flow cytometry following apilimod treatment. Briefly, cells were washed twice with PBS at each time point and whole cell fluorescence was recorded with the Beckman Coulter Cytoflex flow cytometer (Beckman). A total of 10,000 events was counted per condition per sample using the fluorescein isothiocyanate channel for DQ-BSA and LY or phycoerythrin channel for LTR and LAMP1-mCherry and Magic red. Background signal was determined from nonlabeled cells at time 0.

Quantitative RT PCR
RNA isolation from MEF cells was performed through Qiagen RNeasy mini kit (Qiagen). Superscript IV Vilo cDNA synthesis kit (Thermo Fisher) was used to reverse transcribe equal amount of mRNA. The resulting cDNA was amplified through quantitative PCR using TaqMan Fast Advanced Master mix (Applied Biosystems) according to manufacturer instructions in presence of Taqman assays with QuantStudio 3 Real-Time PCR system (Thermo Fisher) controlled by QuantStudio Design and Analysis Software version 1.2 (Thermo Fisher). Taqman assays include Actb (Mm02619580_g1), CtsD (Mm00515586_m1), CtsB (Mm00514443_g1), Atp6v1h (Mm01224350_m1), Atp6v1d (Hs00211133_m1), Lamp1 (Mm01217068_g1), and Mcoln1 (Mm01211241_g1) and were performed in triplicates. Relative quantification (ΔΔCt method) was used to determine gene expression normalized to Actb and vehicle-treated WT MEF.

Statistical analysis
All experiments are conducted independently at least three times. All error bars represent SD. Statistical analysis to compare significance between multiple conditions were performed by ANOVA and post hoc analyses were performed with multiple Student's t test with Bonferroni correction. p values less than 0.05 considered were statistically significant.

Upon Request
Supporting information-This article contains supporting information.